Abstract: Vitamin B3 (niacin) in its functional cofactor forms is found as nicotinamide adenine dinucleotide (NAD+) (phosphate) and reduced forms (referred as NAD(P)(H)). These cofactors participate in more reactions than any other known vitamin-derived molecules and are intimately implicated in essential bioenergetics, anabolic and catabolic pathways. Through its roles in mitochondrial respiration (OXPHOS), reactive oxygen species (ROS) inhibition and additional roles in cellular signalling, the NAD(P)(H) metabolome is central to cellular homeostasis and growth. Acute vitamin B3 deficiency leads to pellagra, a debilitating and deadly disease still endemic in some regions of the world, even in affluent populations. In the US, clinical vitamin B3 deficiency is due to poor food choices, adverse drug reactions, alcoholism and infectious or autoimmune diseases. Sustained sub-optimal dietary intake of vitamin B3 has been shown to have long term physiological consequences, while health benefits associated with vitamin B3 supplementation remain poorly understood. Critically, excessive food intake changes vitamin B3 metabolism and the bioavailability of its functional cofactors, leading to mitochondrial dysfunction and cellular impairment, mainly impacting organs with high metabolic turnover such as liver, kidneys, brain and heart. Often overlooked is that vitamin B3's bioconversion to its functional cofactors depends on the bioavailability of other dietary vitamin Bs' derivatives, in particular thiamine (vitamin B1) and riboflavin (vitamin B2). We hypothesize that a deficiency, even partial, in vitamin B1 and B2 will negatively impact the bioavailability of the vitamin B3's functional forms, even in time of vitamin B3 supplementation, leading to adverse physiological outcomes. We propose to measure and compare the effects that changes in the bioavailability of the functional forms of vitamin B1 and B2, have on the NAD(P)(H) metabolome. Using our synthetic expertise in nucleotide and stable isotope labelling chemistry, we will generate isotopically labelled biosynthetic precursors and intermediates to examine the synergism between the bioavailability of these three vitamins' cofactors and mitochondrial functions under different controlled supplementation conditions in human liver and kidney cells. Further, we will determine if cells compensate for an NAD+ shortage imposed by vitamin B1 and B2 biosynthetic temporary deficiencies by directly generating NAD+ through the Nicotinamide Riboside dependent NAD+ biosynthetic pathway to extend/ resume mitochondrial function. This knowledge will support our long-term goal of identifying new, physiologically relevant vitamin-B combinations which better restore mitochondrial function in cells and organs, where metabolism has been compromised by imbalanced micronutrition. This knowledge will also improve our understanding of the impacts of a global or partial vitamin B deficiency and vitamin B supplementation on organ's functions in relation to malnutrition and over-nutrition.